[HARRIS] A REDUCING ENDO-ENZYME 129 



or with 0*75% NaCl until the water from the hepatic vein is color- 

 less. The organ is then rapidly cut into large pieces from which a 

 good deal of water is allowed to drain away. The pieces are then cut 

 up into much smaller bits and forced into the juice-press in which 

 they are crushed under considerable pressure. A fawn coloured, 

 viscid liquid drips out and is received under toluene. This juice is 

 subsequently ground up with powdered glass and filtered through 

 two layers of cheese cloth to free it from connective-tissue and the 

 debris of blood-vessels, etc. Some preventative of putrefaction must 

 be used, although any such substances may reduce the energy of 

 tissue-respiration. 



V. Description of a Typical Observation. 



Three cubic centimetres of absolutely fresh press-juice, prepared 

 as just described, were shaken in a test-tube with 10 c.c. of 0-05% 

 solution of soluble Prussian blue at room temperature (about 17°C.) 

 The blue colour began to disappear immediately, and in less than a 

 minute, after passing through light blue, light green and greenish 

 grey, the mixture became light grey in colour. No trace of pigment 

 remained. 



When the same volume of boiled juice was used, no decrease in 

 the intensity of the blue colour of the solution was observed at the end 

 of several hours. The reducing activity of the juice was found to 

 diminish somewhat rapidly with time. With a mixture containing 3 c.c. 

 of the press-juice 24 hours old and 10 c.c. of 0*05% Prussian blue 

 solution, it was found that ten minutes elapsed before its colour be- 

 came green-grey, and two hours before it became completely color- 

 less (grey). 



VI. Examination of Possible Fallacies. 



Since the change from the coloured to the leuco condition is the 

 sign of reduction having taken place, one must guard against con- 

 fusing the fading of pigments through reduction with fading from 

 causes other than chemical reduction. 



(a) The earliest criticism offered was that the fading of the Prussian 

 blue was due to the presence of "alkaline salts." Now free alkali, 

 which undoubtedly fades Prussian blue, does not exist in the tissues 

 or their juices. The inorganic salts of tissues and tissue-juices do not 

 bring about any fading of solul)lc Prussian blue. 



Ringer's solution added warm to Prussian blue produces no 

 change of colour beyond that due to a corresponding dilution with 

 water. 



